The long-term objective of the experiments described here is to increase our understanding of HIV-l replication. Recent work suggests that specific binding of HIV-I Gag to cellular proteins known as cyclophilins (CyPs) is necessary for viral infectivity. CyPs are ubiquitous prolyl isomerases thought to function as chaperones. Members of this family of proteins are required for the immunosuppression induced by cyclosporin A (CsA) and are suspected regulators of cellular activation pathways. The overall goal of this project is to determine the significance of the Gag-CyP interaction for HIV- l replication. First, minimal sequence requirements and individual residues important for CyP binding will be determined using a genetic assay (the two-hybrid system) to screen libraries of randomly generated HIV-l gag mutants for the ability to bind to CyP. Subsequently, point mutants which disrupt the Gag-CyP interaction will be used as parents for further mutagenesis intended to identify second-site revertants which restore binding activity. Primary sequence information derived from these studies will be useful for subsequent studies designed to determine the function of the Gag- CyP interaction. The second aim is to determine the range of retroviruses encoding Gags which bind CyP. A panel of gag genes from representative retroviruses will be subcloned into expression vectors for testing in two binding assays. Sequence alignments between these viruses will lead to hypotheses about the importance of particular residues for binding to CyP. These will be confirmed by site-directed mutagenesis and by the construction of gag chimeras. Information obtained from this analysis will provide useful functional probes of the Gag-CyP interaction, and may lead to the development of animal models for its study. The third aim is to identify the essential function that the Gag- CyP interaction serves for HIV- l. Five functional probes will be exploited to maximize correlation between structure and function: l. drugs which block the Gag-CyP interaction; 2. gag mutations which disrupt the Gag-CyP interaction; 3. second-site revertants which restore binding to disruptive gag mutants; 4. a panel of retroviruses encoding Gag proteins with a range of CyP binding affinities; and 5. HIV-l isolates resistant to CsA. First, the viral life cycle as a whole will be examined. Then, a plaque reduction assay employing defective proviruses will be used to distinguish between effects on early and late events. Finally, the importance of the Gag-CyP interaction for each step of the HIV-l life cycle will be systematically examined. Included in this analysis will be a determination of the requirements for the specific incorporation of CyP A into virion particles by Gag. These studies will provide detailed information about basic molecular events necessary for HIV-l infectivity.